I have been using FITC-phalloidin (binds polymerized actin) to look at chemokine responsiveness of human lymphocytes. By titering down the phalloidin, I have been able to measure it on a linear scale and compensate against log PE, so it is a four color functional assay, 3 colors for phenotype and phalloidin. This is done on a FACSCalibur if that makes a difference to those of you considering this problem. My problem is that I am particularly interested in a rare population of cells that is defined by being double positive for two of the markers. Since the increase of phalloidin staining is two or three fold of baseline, doublets are a concern. I thought I was being pretty clever about this by using FL-1 area to exclude doublets, but now I am not so sure. In looking at the analyses with and without the "doublets" excluded I might be losing true positive events. This might be particularly true if polymerization of actin would be expected to affect cell shape, and hence pulse width and area. Is that true? Has anyone ever tried multicolor analyses with phalloidin (or any other probe that one collects on a linear scale). One issue is cell number collected. I try to collect at 3000/second because I need to collect a total of about 600K to analyze all subpopulations of interest. That leaves each collection to about 5-6 minutes. I know if I collect more than 3K/second on a Calibur doublets is an increasing problem. I include this in case it is part of my problem. thanks. ron Ronald L. Rabin, M.D. Research Fellow Laboratory of Clinical Investigation NIAID/NIH Phone: 301-402-4910 FAX: 301-402-0627 Email: rr84g@nih.gov Ronald L. Rabin, M.D. Research Fellow Laboratory of Clinical Investigation NIAID/NIH Phone: 301-402-4910 FAX: 301-402-0627 Email: rr84g@nih.gov
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